Protein allosterism is the basis for the molecular control mechanisms that regulate the fundamental processes of living systems; processes such as the expression of genetic information, cellular metabolism, and the transmission of nerve impulses. Despite the importance of protein allosterism, basic questions concerning the molecular mechanisms of allosterism-questions such as whether or not this phenomena is based mainly on a concerted two-state mechanism-are still intensely debated. Almost all the structural and functional research on allosteric proteins to date has focused on the bulk solution proteins of these proteins (where a complex mixture of ligation intermediates is present), or on the beginning and end states of the ligation pathway (i.e., on only the completely unliganded protein and on the fully liganded protein). A basic premise of this proposal is that a comprehensive set of structural and functional data on the individual ligation states of an allosteric protein is a prerequisite to developing an accurate stereochemical model that fully describes its mechanism of action. In the case of hemoglobin, Ackers and co-worker have taken a large step toward this ambitious goal by determining for the first time a complete set of thermodynamic relationships between all of hemoglobin's ten ligation states. These data revealed a "molecular code mechanism" which specifies that the ligand affinities of the unliganded subunits on a alpha2beta2 hemoglobin tetramer depend on the configuration of the occupied heme sites, not solely on the total number of occupied sites. The central objective of this Program Project is to test and further develop a predictive stereochemical model, formulated during the last funding period, that provides a structural basis for the thermodynamic molecular code mechanism. This goal is now feasible because of major advances (made by Core C of this Program Project) in the development and optimization of chemical cross-linking and heme substitution technologies that allow the eight intermediate ligation states of hemoglobin to be made in large quantities and in pure form. The proposed stereochemical model will be tested and refined by specific mutations experiments and by detailed studies of the ligation intermediates using ligand binding and kinetic measurements, X-ray diffraction methods, Resonance Raman spectroscopy, ENDOR spectroscopy, and advanced hydrogen exchange strategies. This Project brings together six established research groups with complementary areas of expertise and a common interest in understanding the molecular mechanisms of protein allosterism. Work in the different laboratories will be coordinated through planned collaborations, the exchange of materials, and quarterly meetings. This research would result in new insights into hemoglobin's molecular mechanism that may provide for better understanding and treatment of hemoglobinopathies and for the design of improved blood substitutes.